The present invention concerns a composition containing lycopene and Vitamin E.
This present invention more particularly relates to a synergistic mixture of lycopene and vitamin E and its use in the prevention of LDL oxidation, and preparing pharmaceutical and dietary compositions for arresting the progression of atheroclerosis.
Atherosclerosis is the major single cause of mortality in western society. It is assumed to reach up to 50% of all mortality (Davies M J, and Woolf N. Atherosclerosis: what is it and why does it occur? Br Heart J 69:S3, 1993). In addition, it results in significant cardiac morbidity, such as anginal syndromes, myocardial infarctions, ischemic cardiomyopathy, sudden cardiac death, cerebrovascular accidents, and peripheral vascular disease. Indisputable evidence for an association between coronary heart disease (CHD) and risk factors, such as arterial hypertension, cigarette smoking and hyperlipidemia, has been derived from a variety of epidemiological studies. Of all the risk factors established thus far, lipid disorders play a key role in the pathogenesis of atherosclerotic vascular disease, especially of CHD. Many epidemiological and clinical trials have demonstrated the powerful association between hyperlipidemia and the widespread incidence of CHD. The Framingham Heart Study (Castelli W P, Anderson K, Wilson P W., Levy D. Lipids and risk of coronary heart disease. The Framingham Study. Ann Epidemiol 2(1-2): 23-28, 1992), which has been continuous since 1984, showed that hypercholesterolemia is a major contributor to the development of CHD. The link between atherosclerosis and cholesterol has been confirmed by a number of clinical trials.
Fats are insoluble in the aqueous medium of the blood. Thus, transport of the lipids triglycerides, phospholipids and cholesterol occurs exclusively by way of lipid-protein complexes, the lipoproteins. The lipoproteins are classified into 4 broad classes-chylomicrons, very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein (HDL), which differ in their composition, size, and potential atherogenicity.
Measurement of total cholesterol level in plasma reflects the sum of cholesterol being transported in each individual lipoprotein. However, LDL and HDL are the main cholesterol carriers in plasma, and only a small fraction of cholesterol is carried in VLDL or in the chylomicrons. Atherosclerosis is a gradual pathological process, which is characterized by an accumulation of lipid filled macrophages (foam cells), and smooth muscle cells resulting in lesions that thicken and harden the arterial wall. The main source for the cholesterol accumulating in the foam cells is the circulating LDL. There is evidence from numerous epidemiological and clinical studies that LDL, as the carrier of ca. 70% of the total cholesterol in plasma, are the most potent atherogenic lipoproteins. Their elevation carries a particular risk, and reduction in LDL cholesterol constitutes a diminished atherosclerotic risk. The Lipid Research Clinics Coronary Primary Prevention Trial (LRC-CPPT) had for the first time presented firm proof that in man, a lowering of LDL cholesterol level reduces the rate of myocardial infarcaiton and infarction mortality.
Nearly all cells, including macrophages, take up exogenous cholesterol via LDL-receptors. Increase in cell cholesterol content, however, results in down-regulation of LDL receptor number, thereby protecting cells from excessive accumulation of cholesterol by way of this pathway. It was shown that chemical or biological modification, including oxidation of LDL, results in increased uptake of the modified lipoprotein by way of other cell surface receptors, termed scavenger receptors. These receptors are present on macrophages and endothelial cells.
Oxidative modification of LDL is thought to play a causal role in atherosclerosis: (see e.g. Steinberg D., Parthasarathy S., Carew T. E., Khoo J. C. and Witztum J. L, xe2x80x9cBeyond cholesterol: modifications of low-density lipoprotein that increase its atherogenicityxe2x80x9d, N. Engl. J. Med. 1989; 320: 915-924; Haberland M. E. and Fogelman A. M., xe2x80x9cThe role of altered lipoproteins in the pathogenesis of atherosclerosisxe2x80x9d, Am. Heart. J. 1987; 113: 573-577; and Witztum J. L., xe2x80x9cThe oxidation hypothesis of atherosclerosisxe2x80x9d, Lancet. 1994; 344, 793). It is believed, accordingly, that prevention of LDL oxidation by antioxidants may arrest the progression of atherosclerosis. Aviram M. Beyond cholesterol: Modification of lipoproteins and increased atherogenicity. In Atherosclerosis, inflammation and thrombosis (G. G. Neri Serneri, G. F. Gensini, R. Abbate and D. Prisco eds)-Scientific Press-Florence, Italy, pp: 15-36, 1993.
The ability to prevent the development of atherosclerotic lesions would have major implications for the public health. Thus using therapeutic agent with plural effects, such as lowering cholesterol and inhibiting oxidative modification, might have beneficial effects over other individual agent.
Carotenoids are colored pigments with lipophilic properties, widely distributed in fruits and vegetables, (e.g. xcex2-carotene in carrots and lycopene in tomatoes) and possess some antioxidant properties: (see e.g. Burton G. W., xe2x80x9cAntioxidant action of carotenoids,xe2x80x9d 1989; J. Nutr. 119:109-111 and Krinsky N. I., xe2x80x9cAntioxidant functions of carotenoidsxe2x80x9d, Free Radic. Biol. Med. 1989, 7: 617-635 (9-13)). Carotenoids are transported within circulating lipoproteins, and it was postulated that they participate in the protection of LDL from oxidative modification.
Carotenoids consumption was shown in previous epidemiological studies to be associated with reduced cardiovascular mortality (see e.g.: Kohlmeier L. and Hasting S. B., xe2x80x9cEpidemiologic evidence of a role of carotenoids in cardiovascular disease preventionxe2x80x9d, Am. J. Clin. Nutr. 1995; 62: 137S-146S), although recent data did not demonstrate similar beneficial effect (see e.g.: Hennekens C. H., Buring J. E., Manson J. E., Stampfer M, Rosier B., Cook N. R., Belanger C., LaMotte F., Gaziano J. M., Ridker P. M., Willett W. and Peto R., xe2x80x9cLack of effect of long-term supplementation with beta carotene on the incidence of malignant neoplasms and cardiovascular diseasexe2x80x9d, N. Engl. J. Med. 1996; 334: 1145-1149).
Reduced plasma lipid peroxidation was recently shown to be associated with increased consumption of tomatoes. Low levels of plasma carotenoids were shown to be associated with an increased risk of myocardial infarction, and recently it was demonstrated that the association between xcex2-carotene and acute myocardial infarction depends on the polyunsaturated fatty acids status, and that feeding the all-trans isomer of xcex2-carotene to cholesterol-fed rabbits attenuated the extent of their atherosclerosis, with no effect on LDL oxidizability ex vivo. Data on the ability of xcex2-carotene supplementation in vitro or in vivo to protect LDL from oxidation are conflicting: some studies found an inhibitory effect of xcex2-carotene supplementation on LDL oxidation, whereas several other did not find such effect.
Lycopene, the open chain analog of xcex2-carotene, shares with it similar structure with an extended conjugated double bonds. In human plasma, lycopene and xcex2-carotene are quantitatively the major carotenoids. Lycopene was shown to possess the greatest quenching ability of singlet oxygen among the various carotenoids (DiMascio P., Kaiser S. and Sies H., xe2x80x9cLycopene as the most efficient biological carotenoid singlet oxygen quencher,xe2x80x9d Arch. Biochem. Biophys., 1989, 274: 532-538) and it was shown to be at least twice as effective antioxidant as xcex2-carotene in protecting blood lymphocytes form NO2 radical damage (Bohm F., Tinkler J. H., and Truscott T. G., xe2x80x9cCarotenoids protect against cell membrane damage by the nitrogen dioxide radicalxe2x80x9d, Nature Medicine, 1995, 2: 98-99).
We have recently demonstrated a protective effect of tomatoes lycopene against oxidative modification of LDL. This protection of LDL by lycopene exceeds the protection exhibited by xcex2-carotene, was selective only to LDL""s with high vitamin E content and was shown when the carotene was present in combination with vitamin E. (see e.g., Fuhrman B, Ben Yaish L, Attias J. Hayek T. Aviram M. Tomatoes lycopene and xcex2-carotene inhibit low density lipoprotein oxidation and this effect depends on the lipoprotein vitamin E. content. Nutr Metab. Cardiovasc. Dis 7: 433-443, 1997). Furthermore, we have also demonstrated that dietary supplementation of lycopene acts as moderate hypocholesterolemic agent secondary to its inhibitory effect on cellular cholesterol syntheses (see e.g. Fuhrman B., Elis A., Aviram M., Hypocholesterolemic effect of lycopene and xcex2-carotene is related to suppression of cholesterol synthesis and augmentation of LDL receptor activity in macrophages. Bichem. Biophys. Res. Commun. 233: (658-662, 1997).
Israel Patent Application No. 123,132 recently filed covers the use of a mixture of lycopene and garlic to be used in preventing LDL oxidation.
It is an objective of the present invention to provide a novel mixture of lycopene and vitamin E. It is an objective of the present invention to provide a synergistic mixture of lycopene and vitamin E active in blocking the oxidation of LDL and/or atherosclerosis and reduce the cholesterol levels in plasma. An additional objective of the present invention is the use of the above described mixture to prepare a pharmaceutical or dietary composition for arresting the progression of atherosclerosis. A further objective of the present invention is to provide a composition for the arresting of the progression of atherosclerosis containing essentially products acceptable and desertable of the harm diet.
The invention is based on the discovery that there is an unexpected and surprising synergistic effect between lycopene and vitamin E, in the prevention of LDL oxidation. Lycopene is a natural product and is present in tomatoes in various concentrations, but can also be produced by fungal, algal or by fermentation, in genetically modified organisms (GMO) or by synthetic method. Compositions comprising it and their use are comprised in the present invention. A widely used source of vitamin E is soybean distillate.
The invention therefore provides a composition which is a combination of lycopene and vitamin E.
The composition may contain other dietary components, additives, excipients, binding agents, coatings, etc., or other compounds that have no significant effect in preventing LDL oxidation. The compositions according to the invention, in view of their purpose, are pharmaceutical compositions. However, since their components are individually acceptable ingredients of the human diet, and in fact are individually present in foodstuffs, they may be considered and used as dietary or health supplements.
The use of the aforesaid combinations of compounds as medicines or components of pharmaceutical compositions or of dietary or health compositions for the prevention of LDL oxidation is also an aspect of the present invention.
The use of tomato oleoresin for the prevention of LDL oxidation is a specific aspect of the present invention.
The compositions according to the invention should preferably comprise lycopene and vitamin E in a molar proportion of 1:400 to 10:1, preferably 1:80 to 5:1. The amount of lycopene in each unit of the composition, e.g. a tablet or soft gel capsule, should be in the range of 1-4, preferably about 2-3 and up to 15 mg of lycopene.
The invention comprises a method of preventing LDL oxidation, and therefore arresting the progression of atherosclerosis, which comprises administering to a patient lycopene and vitamin E, in the aforesaid relative amounts.
In a preferred way of carrying out the invention, and in the Examples that will be described, lycopene was obtained from tomato oleoresin. The oleoresin was supplied by LycoRed-Natural Products Industries, Ltd, Beer Sheva, Israel. It consisted of crystalline lycopene (6.0%) suspended in the lipid phase of the tomato. Fatty acids as triglycerides constituted the major part (72%) of the oleoresin, whereas 19% were non-saponified materials including 6.0% lycopene, 0.1% xcex2-carotene and 1% vitamin E. The rest consists of water and water soluble materials. The lycopene used was extracted and purified mainly in the all-trans configuration. Lycopene can be extracted from tomato oleoresin, as described in Patent Application No. WO 97/48287.
Vitamin E is readily available in the pharmaceutical market.
Said ingredients are conveniently available and used in the form of oleoresin or in a solid form such as beadlets.
Materials
Supplementation of Human LDL with Lycopene
Stock solutions of 1 mM of purified lycopene or of 10 mg/ml of tomato""s oleoresin, were prepared in Tetrahydrofuran (THF, HPLC grade). All procedures were performed in dim light. LDL was incubated with the respective concentrations of tomato""s oleoresin or with 3 xcexcM of lycopene (3 xcexcl/ml THF, derived from a stock solution of 1 mM) for 30 minutes at 37xc2x0 C. in the dark. LDL incubated with THF alone served as control.
LDL Oxidation
Oxidation of LDL was carried out in a shaking water bath at 37xc2x0 C. under air, in plastic tubes (1 cm in diameter). For metal ions dependent oxidation LDL was incubated for 4 hours at 37xc2x0 C. with freshly prepared CuSO4 (5 xcexcM).
LDL oxidation was determined by measuring the amount of thiobarbituric acid reactive substances (TBARS) (See: Buege J. A. and Aust S. D. Microsomal lipid peroxidation. Methods Enzymol. 1978; 52: 302-310) and by lipid peroxides formation using a commercially available kit.
Effects of Lycopene or Vitamin E Alone Against LDL Oxidation
LDL (100 xcexcg of protein/ml) was preincubated for 30 min at 37xc2x0 C. with either vitamin E or lycopene in various forms. LDL oxidation was induced by the addition of 5 xcexcM of CuSO4 and was preincubated for 30 min at 37xc2x0 C. LDL oxidaton was measured by the TBARS assay or by measuring the inhibition of the formation of lipid peroxides. Results are shown in FIG. 1 for vitamin E inhibition and FIG. 2 for crystalline lycopene. FIG. 3 shows the effect for tomato oleoresin, and for comparison, FIG. 4 shows the results for tomato oleoresin where the natural occurring vitamin E was extracted out.
Effect of Lycopene in Combination With Vitamin E Against LDL Oxidation
The antioxidative effect of a combination of vitamin E with lycopene against LDL oxidation, was assessed is a manner illustrated by FIGS. 5 to 7, in which the degree of inhibition of LDL oxidation by individual anti-oxidants is shown in the ordinate.
The tests hereinbefore described show that when vitamin E and lycopene are added together to LDL during copper-induced oxidation, a synergistic effect occurs. This synergistic inhibitory effect on LDL oxidation, is believed to be probably related to the contribution of vitamin E and lycopene to different antioxidative mechanisms. Whereas vitamin E is a chain breaking agent and acts as a free radicals scavenger, lycopene is a potent free radical scavenger as well as an oxygen quencher. Therefore, administration of a combination of these two antioxidants with synergistic inhibitory effects on LDL oxidation, such as vitamin E and lycopene, appears to be beneficial over the administration of individual antioxidants separately. This is further evidenced by the inhibitory effect on LDL oxidation of tomato""s oleoresin, which contains a combination of antioxidants.
In some cases as published in the literature, vitamin E alone does not efficiently lower the LDL oxidation. Nevertheless, when lycopene or the oleoresin of tomatoes is added to the vitamin E, a marked synergistic lowering of the LDL oxidation occurs.